Tissue inhibitor of metalloproteinase type three (TIMP-3) composition and methods

ABSTRACT

The present invention relates in general to metalloproteinase inhibitors and to polynucleotides encoding such inhibitors. In particular, the invention relates to novel mammalian inhibitors of metalloproteinase, which are designated as type three or TIMP-3, to fragments, derivatives, and analogs thereof, and to polynucleotides encoding the same. Novel methods of producing such compositions and novel methods of using such compositions are also provided.

This is a division of application Ser. No. 08/134,231, filed Oct. 6,1993, now U.S. Pat. No. 6,562,596 which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates in general to metalloproteinase inhibitorsand to polynucleotides encoding such factors. In particular, theinvention relates to novel mammalian tissue inhibitors ofmetalloproteinase (herein designated as type three, or “TIMP-3”), tofragments, derivatives, and analogs thereof and to polynucleotidesencoding the same. In another aspect, the present invention relates tonovel methods of producing such compositions, and methods of using suchcompositions.

BACKGROUND OF THE INVENTION

Connective tissues are maintained in dynamic equilibrium by the opposingeffects of extracellular matrix synthesis and degradation. Theextracellular connective tissue matrix consists predominantly ofcollagens, with proteoglycans, fibronectin, laminin and other minorcomponents making up the remainder.

Degradation of the matrix is brought about by the release of neutralmetalloproteinases from resident connective tissue cells and invadinginflammatory cells that are capable of degrading at physiological pHmost of the matrix macromolecules. See Table 1, below. The proteinasesinclude the mammalian tissue collagenases, gelatinases, andproteoglycanases; leukocyte collagenase and gelatinase (Murphy et al.Biochem. J. 283: 289–221 (1982); Hibbs et al., J. Biol. Chem. 260:2493–2500 (1985)); macrophage collagenase and elastase (Werb et al. J.360 (1975); Banda et al., Biochem. J. 193: 589–605 (1981)); and tumourcollagenases (Liotta et al., PNAS-USA 76: 2268–2272 (1979); Liotta etal., Biochem. Biophys. Res. Commun. 98: 124–198 (1981); and Salo et al.,J. Biol. Chem. 258: 3058–3063 (1983)). For a general review ofcollagenases and their role in normal and pathological connective tissueturnover see Collagenase in Normal and Pathological Connective Tissues,David E. Woolley and John M. Evanson, eds., John Wiley & Sons Ltd.(1988).

There are over five different collagen types (I, II, III, IV, V, etc.)which are differentially distributed among tissues. There isconsiderable homology and structural similarity among the variouscollagen types. Particular collagenases show some specificity forparticular collagen types. See Table 1, below; Matrisian, Trends InGenetics 6: 121–125 (1990). With regard to inhibition of collagenasesand other matrix-degrading metalloproteinases, it is possible that,depending on the actual enzymes, substrates, and inhibitory mechanisms,an inhibitor could act on just one, on several, or on all collagenasesand metalloproteinases.

TABLE 1 MATRIX-degrading metalloproteinases Name(s) Size (kDa) DegradesRef. (1) Interstitial collagenase 52 deduced I, II, III collagen Scholtzet al., Cancer Res. 48: 5539– (Type I collagenase) 52, 57 secreted 5545(1988) (MMP-1) PMN Collagenase 75 secreted I, II, III collagen Macartneyet al., Evr. J. Biochem. (MMP-8) 130: 71–78 (1983). (2) 72 kDA Type IVcollagenase 72 secreted IV, V, VII collagen, Collier et al., J. Biol.Chem. (72 kDa gelatinase) fibronectin, gelatins 263: 6579–6587 (1988)(MMP-2) 92 kDa Type IV collagenase 78 deduced IV, V collagen, gelatinsWithelm et al., J. Biol. Chem. 263: (92 kDa gelatinase) 92 secreted17213–17221 (1989) (MMP-9) (3) Stromelysin 53 deduced Proteoglycans,laminin, fibronectin, Chin et al., J. Biol. Chem. 260: (transin) 57, 60secreted III, IV, V collagen, gelatins 12367–12376 (1985)(proteoglycanase) (procollagen-activiating factor) (MMP 3) Stromelysin-253 deduced III, IV, V collagen, fibronectin, Nicholson et al.,Biochemistry 28: (transin-2) gelatins 5195–5203 (1989) (MMP-10) PUMP-128 deduced Gelatins, fibronectin Quantin et al., Biochemistry 28:(MMP-7) 28 secreted 5327–5333 (1989) (Small metalloproteinase of uterus)The matrix metalloproteinases are divided into three major subclasses,indicated with arabic numerals, on the basis of their substratespecificities. The enzymes in each class are bold, and alternative namesare shown in parentheses. MMP, matrix metalloproteinase; PMN,polymorphonuclear leukocyte.

The underlying basis of degradative diseases of connective tissue pointsto the matrix-specific metalloproteinases as having a fundamental rolein the etiology of these diseases. Such diseases include dystrophicepidermolysis bullosa; rheumatoid arthritis; corneal, epidermal orgastric ulceration; peridontal disease; emphysema; bone disease; andtumor metastasis or invasion.

Most studies on connective tissue degradation and diseases involvingsuch degradation have limited the measurement of metalloproteinases tocollagenase (the most widely studied of this group ofmetalloproteinases). It is understood however, that the simultaneouseffects of collagenase and the other matrix-degrading metalloproteinaseswill exacerbate the degradation of the connective tissue over thatachieved by collagenase alone.

Specific natural inhibitors of collagenase were discovered in crudemedium from cultured connective tissues. A metalloproteinase inhibitorknown as TIMP (tissue inhibitor of metalloproteinases) has been studiedwith regard to physicochemical properties and the biochemistry of itsinteraction with collagenase, Murphy et al., J. Biochem. 195: 167–170(1981); Cawston et al., J. Biochem. 211: 313–318 (1983); Stricklin etal., J. Biol. Chem. 258: 12252–12258 (1983), and DNA encoding it hasbeen isolated, Docherty et al., Nature 318: 65–69 (1985); Carmichael etal., PNAS-USA 83: 2407–2411 (1986). In an in vitro cell culture model oftumor cell migration through a natural basement membrane, TIMP was ableto arrest migration of a collagenase-secreting tumor cell line,Thorgeirsson et al., J. Natl. Canc. Inst. 69: 1049–1054 (1982). In vivomouse lung colonization by murine B16-FlO melanoma cells was inhibitedby injections of TIMP, Schultz et al., Cancer Research 48: 5539–5545(1988). European Patent Publication No. EP O 189 784 also relates toTIMP.

McCartney et al., Eur. J. Biochem. 130: 79–83 (1983) reported thepurification of a metalloproteinase inhibitor from human leukocytes.

DeClerck et al., Cancer Research 46: 3580–3586 (1986) described thepresence of two inhibitors of collagenase in conditioned medium frombovine aortic endothelial cells.

Murray et al., J. Biol. Chem. 261: 4154–4159 (1986) reported thepurification and partial amino acid sequence of a bovinecartilage-derived collagenase inhibitor.

Langley, et al. EP O 398 753 (“Metalloproteinase Inhibitor,” publishedNov. 22, 1990) discloses a novel metalloproteinase inhibitor andanalogs, polynucleotides encoding the same, methods of production,pharmaceutical compositions, and methods of treatment. The polypeptideof FIG. 2 therein has been referred to as TIMP-2, designating a moleculedistinct from TIMP-1, supra. EP O 398 753 describes both bovine andhuman recombinant TIMP-2.

Staskus et al., J. Biol. Chem. 266: 449–454 (1991) reports a 21 kDaavian metalloproteinase inhibitor obtained from chicken fibroblasts. Theauthors note the biochemical similarities with other members of the TIMPand TIMP-2 group of proteins and state that the avian material may be aTIMP variant or may represent a third protein within themetalloproteinase inhibitor family. (This material is referred to hereinas “ChIMP-3”)

Pavloff et al., J. Biol. Chem. 267: 17321–17326 (1992) discloses thecDNA and primary structure of a metalloproteinase inhibitor from chickenembryo fibroblasts.

Yang et al., PNAS-USA 89: 10676–10680 (1992) reports on the role of a 21kDa protein chicken TIMP-3.

The present work relates to a third type of metalloproteinase inhibitorpolypeptides. In one aspect, the present invention involves the cloningof recombinant human TIMP-3 nucleic acid and expression thereof.

SUMMARY OF THE INVENTION

According to the present invention, a class of novel tissue inhibitorsof metalloproteinase are provided. For convenience, the presentpolypeptides are referred to as “TIMP-3,” as these polypeptidesrepresent a new class of members of the tissue inhibitors ofmetalloproteinases. Also provided are DNA sequences coding for all orpart of the present TIMP-3's, vectors containing such DNA sequences, andhost cells transformed or transfected with such vectors. Alsocomprehended by the invention are methods of producing recombinantTIMP-3's, and methods of treating disorders. Additionally,pharmaceutical compositions including TIMP-3's and antibodiesselectively binding TIMP-3's are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–B show the cDNA sequence (SEQ ID NO:12) an animo acid sequence(SEQ ID NO:13) of a recombinant human tissue inhibitor ofmetalloproteinase type 3 (“TIMP-3”). The entire 1240 base pair sequenceencoding a full-length polypetide of 211 amino acids is presented. Ahydrophobic leader sequence is found at position −23 to −1. The initialcysteine of the mature protein is numbered +1. The amino acidscorresponding to the degenerate oligonucleotides which identified theoriginal PCR products are underlined, except that the oligocorresponding to YTIK (SEQ ID NO:36) was used analytically to confirmthe identity of the PCR products prior to sequencing. A potentialglycosylation site is italicized. A variant polyadenylation signalsequence is marked with asterisks. (The abbreviations are used hereinfor amino acids, either single letter or triple letter abbreviations,and nucleic acids are those conventionally used, as in Stryer,Biochemistry, 3d ed. 1988, W. H. Freeman, N.Y., inside back cover.)

FIG. 2 is a photograph of an agarose gel of first-strand PCR products,which demonstrates amplification of human nucleic acid. Lane 1 presentsPCR products from human fetal kidney cDNA primed with primers 449-15(SEQ ID NO:1) and 480-27 (SEQ ID NO:2). Lane 2 presents the results ofPCR amplification of fetal kidney first strand cDNA primed with primers449-15 (SEQ ID NO: 1) and 480-28 (SEQ ID NO: 3). Lane 3 is the PCR kit(Perkin-Elmer-Cetus) control. Lane 4 is TIMP-2 DNA primed with primers449-15 (SEQ ID NO:1)and 480-27 (SEQ ID NO:2). Lane 5 is molecular weightmarkers.

FIG. 3 is a photograph of a silver stained SDS-PAGE gel containingmaterial as follows: Lane 1, molecular weight markers; lane 2, TIMP-2,reduced; lane 3, blank; lane 4, E. coli derived TIMP-3 of FIG. 1,reduced, post-dialysis; lane 5, E. coli derived TIMP-3 of FIG. 1,reduced, post-dialysis, lanes 6, 7, 8, blank; lane 9, E. coli derivedTIMP-3 of FIG. 1, unreduced, pre-dialysis; lane 10, E. coli derivedTIMP-3 of FIG. 1, unreduced, post-dialysis.

FIGS. 4A–C show a comparison of the human TIMP-3 amino acid sequence ofFIG. 1 with other TIMP family members. The numbering begins with thefirst cysteine of the mature protein. As can be seen, the alignmentcontains gaps for some TIMP family members. The numbering used here isconsistent for the numbering used for the recombinant human TIMP-3 ofFIGS. 1A–B. Boldface letters indicate conserved amino acids; asterisksrepresent potential glycosylation sites of TIMP-1; underlined lettersindicate potential glycosylation sites of TIMP-3; the left bracketsindicate the beginning of the mature proteins. A bullet (•) indicatesthose amino acids which are unique to recombinant human TIMP-3. Theamino acid sequences were found in the literature as follows: BovineTIMP-1, Freudenstein et al., Biochem. Biophys. Res. Comm. 171: 250–256(1990); Human TIMP-1, Docherty et al., Nature 318: 65–69 (1985); RabbitTIMP-1, Horowitz et al., J. Biol. Chem. 264: 7092–7095 (1989); MouseTIMP-1, Edwards et al., Nucleic Acid. Res. 14: 8863–8878 (1986); Johnsonet al., Mol. Cell. Biol. 7: 2821–2829 (1978); Gewert et al., EMBO 6:651—651–657 (1987); Bovine TIMP-2, Boone et al., PNAS-USA 87: 2800–2804(1990); Human TIMP-2, Boone et al, PNAS-USA 87: 2800–2804 (1990); MouseTIMP-2, Shimizu et al., Gene 114: 291–292 (1992); Chicken TIMP-3,Pavloff et al., J. Biol. Chem. 267: 17321–17326 (1992). Unless otherwiseindicated, these sequences referred to from time to time herein werefound in these references.

FIG. 5 is a comparison table of the amino acid sequence for the chickenmetalloproteinase inhibitor of Staskus et al., J. Biol. Chem. 266:449–454 (1991) and the recombinant human TIMP-3 of FIG. 1. A solid linebetween amino acids indicates identity, double dots indicatessimilarity. A single dot indicates a lesser degree of similarity, and nodot indicates total difference, as described by Grivskov et al., Nucl.Aud. Res. 14: 6745–6763 (1986).

FIGS. 6A–D show the overall homology between the nucleic acid sequenceencoding TIMP-3 shown in FIGS. 1A–B and that encoding ChIMP-3.

FIGS. 7A–C show the maximal homology between the nucleic acid sequenceencoding TIMP-3 shown in FIGS. 1A–B and that encoding ChIMP-3.

FIG. 8 shows the amino acid sequence alignment of human recombinantTIMP-3 of FIG. 1 and human TIMP-2.

FIGS. 9A–E show the overall homology of the nucleic acid sequence ofhuman recombinant TIMP-3 shown in FIGS. 1A–B and that encoding humanTIMP-2.

FIGS. 10A–C show the maximal homology regions of the nucleic acidsequence encoding human recombinant TIMP-3 shown in FIGS. 1A–B and thatencoding human TIMP-2.

FIG. 11 shows the amino acid sequence alignment of human recombinantTIMP-3 of FIG. 1 and human TIMP-1.

FIGS. 12A–D show the overall homology of the nucleic acid sequenceencoding human recombinant TIMP-3 shown in FIGS. 1A–B and that encodinghuman TIMP-1.

FIG. 13 shows the maximal homology regions of the FIG. 1 nucleic acidsequence encoding human recombinant TIMP-3 and that encoding humanTIMP-1.

FIGS. 14A and B shows Northern blot analyses performed on RNAs from avariety of cells, using a TIMP-3 DNA fragment as a probe.

FIG. 15 shows a modified zymogram. Lane 1 (from the left hand side)contains a protein molecular weight standard (see FIG. 3). Lane 2 is acontrol lane containing conditioned medium with collagenases (72 kDa andinterstitial collagenases, pAPMA activated). (“Coll” refers tointerstitial collagenase.) Lane 3 contains TIMP-2. Lane 4 contains aTIMP-2 analog lacking the six C-terminal cysteines. Lanes 5, 6, and 7contain E. coli derived TIMP-3 of FIG. 1, lane 5 being undiluted andlanes 6 and 7 being consecutive 2-fold serial dilutions. As can be seen,the lack of a clear zone at the location where the control (lane 2)showed clearing indicates that TIMP-3 inhibits collagenase activity.

FIGS. 16A–H show the cDNA and amino acid sequence of variants obtainedusing the present method.

FIG. 17 shows an illustration of a proposed secondary structure ofmembers of the TIMP family of proteins.

Numerous aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the following detaileddescription which provides illustrations of the practice of theinvention in its presently preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, novel metalloproteinase inhibitors(herein called, collectively, TIMP-3) and DNA sequences coding for allor part of such TIMP-3 are provided. Such sequences include theincorporation of codons “preferred” for expression by selectednonmammalian hosts; the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences which facilitate construction of readilyexpressed vectors. The present invention also provides DNA sequencescoding for polypeptide analogs or derivatives of TIMP-3 which differfrom naturally-occurring forms in terms of the identity or location ofone or more amino acid residues (i.e., deletion analogs containing lessthan all of the residues specified for TIMP-3; substitution analogs,wherein one or more residues specified are replaced by other residues;and addition analogs wherein one or more amino acid residues is added toa terminal or medial portion of the polypeptide) and which share some orall the biological properties of mammalian TIMP-3.

Novel nucleic acid sequences of the invention include sequences usefulin securing expression in procaryotic or eucaryotic host cells ofpolypeptide products having at least a part of the primary structuralconformation and one or more of the biological properties of recombinanthuman TIMP-3. The nucleic acids may be purified and isolated, so thatthe desired coding region is useful to produce the present polypeptides,for example, or for diagnostic purposes, as described more fully below.DNA sequences of the invention specifically comprise: (a) the DNAsequence set forth in FIG. 1 (and complementary strands); (b) a DNAsequence which hybridizes (under hybridization conditions disclosed inthe cDNA library screening section below, or equivalent conditions ormore stringent conditions) to the DNA sequence in FIG. 1 or to fragmentsthereof; and (c) a DNA sequence which, but for the degeneracy of thegenetic code, would hybridize to the DNA sequence in FIG. 1. Alsocontemplated are fragments of (a), (b) or (c) above which are at leastlong enough to selectively hybridize to human genomic DNA encodingTIMP-3, under conditions disclosed for the cDNA library screening,below. Specifically comprehended in parts (b) and (c) are genomic DNAsequences encoding allelic variant forms of human TIMP-3 and/or encodingTIMP-3 from other mammalian species, and manufactured DNA sequencesencoding TIMP-3, fragments of TIMP-3, and analogs of TIMP-3 which DNAsequences may incorporate codons facilitating transcription andtranslation of messenger RNA in microbial hosts. Such manufacturedsequences may readily be constructed according to the methods of Altonet al., PCT published application WO 83/04053.

Genomic DNA encoding the present TIMP-3's may contain additionalnon-coding bases, or introns, and such genomic DNAs are obtainable byhybridizing all or part of the cDNA, illustrated in FIGS. 1 and 16, to agenomic DNA source, such as a human genomic DNA library. Such genomicDNA will encode functional TIMP-3 polypeptide; however, use of the cDNAsmay be more practicable in that, since only the coding region isinvolved, recombinant manipulation is facilitated.

According to another aspect of the present invention, the DNA sequencesdescribed herein which encode TIMP-3 polypeptides are valuable for theinformation which they provide concerning the amino acid sequence of themammalian protein which have heretofore been unavailable. Put anotherway, DNA sequences provided by the invention are useful in generatingnew and useful viral and circular plasmid DNA vectors, new and usefultransformed and transfected procaryotic and eucaryotic host cells(including bacterial and yeast cells and mammalian cells grown inculture), and new and useful methods for cultured growth of such hostcells capable of expression of TIMP-3 and its related products.

The DNA provided herein (or corresponding RNAs) may also be used forgene therapy for, example, treatment of emphysema. For example,transgenic mice overexpressing collagenase exhibit symptoms pulmonaryemphysema, D'Armiento et al., Cell 71: 955–961 (1992), indicating thatinhibition of collagenase may ameliorate some of the symptoms ofemphysema. Currently, vectors suitable for gene therapy (such asretroviral or adenoviral vectors modified for gene therapy purposes andof purity and pharmaceutical acceptability) may be administered fordelivery into the lung. Such vectors may incorporate nucleic acidencoding the present polypeptides for expression in the lung.Additionally, one may use a mixture of such vectors, such as thosecontaining genes for one or more TIMPs, elastase inhibitors or otherproteins which ameliorate the symptoms of emphysema. Gene therapy mayinvolve a vector containing more than one gene for a desired protein.

Alternatively, one may use no vector so as to facilitate relativelystable presence in the host. For example, homologous recombination mayfacilitate integration into a host genome. The nucleic acid may beplaced within a pharmaceutically acceptable carrier to facilitatecellular uptake, such as a lipid solution carrier (e.g., a chargedlipid), a liposome, or polypeptide carrier (e.g., polylysine). A reviewarticle on gene therapy is Verma, Scientific American, November 1990,pages 68–84 which is herein incorporated by reference.

As mentioned above, target cells may be within the lungs of therecipient, but other target cells may be bone marrow cells, blood cells,liver (or other organ) cells, muscle cells, fibroblasts, or other cells.The desired nucleic acid may be first placed within a cell, and the cellmay be administered to a patient (such as a transplanted tissue) or thedesired nucleic acid may be administered directly to the patient foruptake in vivo.

The cells to be transferred to the recipient may be cultured using oneor more factors affecting the growth or proliferation of such cells, asfor example, SCF.

Administration of DNA of the present invention to the lung may beaccomplished by formation of a dispersion of particles, or an aerosol.Typically some type of bulking agent will be involved, and a carrier,such as a lipid or polypeptide. These materials must be pharmaceuticallyacceptable. One may use a nebulizer for such delivery, such anultrasonic or dry powder nebulizer. Alternatively, one may use apropellant based system, such as a metered dose inhaler, which maydeliver liquid or a suspension of particles.

For gene therapy dosages, one will generally use between one copy andseveral thousand copies of the present nucleic acid per cell, dependingon the vector, the expression system, the age, weight and condition ofthe recipient and other factors which will be apparent to those skilledin the art.

DNA sequences of the invention are also suitable materials for use aslabeled probes in isolating human genomic DNA encoding TIMP-3, asmentioned above, and related proteins as well as cDNA and genomic DNAsequences of other mammalian species. DNA sequences may also be usefulin various alternative methods of protein synthesis (e.g., in insectcells) or, as described above, in genetic therapy in humans and othermammals. DNA sequences of the invention are expected to be useful indeveloping transgenic mammalian species which may serve as eucaryotic“hosts” for production of TIMP-3 and TIMP-3 products in quantity. See,generally, Palmiter et al., Science 222: 809–814 (1983).

Also, one may prepare antisense nucleic acids against the present DNAs.Compare, Khokho et al., Science 243: 947–950 (1989), whereby antisenseRNA inhibitor of TIMP conferred oncogenicity on Swiss 3T3 cells.Antisense nucleic acids may be used to modulate or prevent expression ofendogenous TIMP-3 nucleic acids.

The present invention provides purified and isolated polypeptideproducts having part or all of the primary structural conformation(i.e., continuous sequence of amino acid residues) and one or more ofthe biological properties (e.g., immunological properties and in vitrobiological activity) and physical properties (e.g., molecular weight) ofnaturally-occurring mammalian TIMP-3 including allelic variants thereof.The term “purified and isolated” herein means substantially free ofunwanted substances so that the present polypeptides are useful for anintended purpose. For example, one may have a recombinant human TIMP-3substantially free of other human proteins or pathological agents. Thesepolypeptides are also characterized by being the a product of mammaliancells, or the product of chemical synthetic procedures or of procaryoticor eucaryotic host expression (e.g., by bacterial, yeast, higher plant,insect and mammalian cells in culture) of exogenous DNA sequencesobtained by genomic or cDNA cloning or by gene synthesis. The productsof expression in typical yeast (e.g., Saccharomyces cerevisiae) orprocaryote (e.g., E. coli) host cells are free of association with anymammalian proteins. The products of expression in vertebrate (e.g.,non-human mammalian (e.g. COS or CHO) and avian) cells are free ofassociation with any human proteins. Depending upon the host employed,and other factors, polypeptides of the invention may be glycosylatedwith mammalian or other eucaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue (at position −1 with respect tothe first amino acid residue of the polypeptide).

In addition to naturally-occurring allelic forms of TIMP-3, the presentinvention also embraces other TIMP-3 products such as polypeptideanalogs of TIMP-3 and fragments of TIMP-3. Following the procedures ofthe above noted published application by Alton et al. (WO 83/04053), onecan readily design and manufacture genes coding for microbial expressionof polypeptides having primary conformations which differ from thatherein specified for in terms of the identity or location of one or moreresidues (e.g., substitutions, terminal and intermediate additions anddeletions). Alternately, modifications of cDNA and genomic genes may bereadily accomplished by well-known site-directed mutagenesis techniquesand employed to generate analogs and derivatives of TIMP-3. Suchproducts would share at least one of the biological properties ofmammalian TIMP-3 but may differ in others. As examples, projectedproducts of the invention include those which are foreshortened by e.g.,deletions; or those which are more stable to hydrolysis (and, therefore,may have more pronounced or longer lasting effects thannaturally-occurring); or which have been altered to delete one or morepotential sites for glycosylation (which may result in higher activitiesfor yeast-produced products); or which have one or more cysteineresidues deleted or replaced by, e.g., alanine or serine residues andare potentially more easily isolated in active form from microbialsystems; or which have one or more tyrosine residues replaced byphenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within TIMP-3, which fragments may possess oneactivity of mammalian TIMP-3 (e.g., immunological activity) and notothers (e.g., metalloproteinase inhibiting activity).

The present TIMP-3 may bind to the extracellular matrix, acharacteristic not shared by TIMP-1 and TIMP-2. Thus, polypeptidesexhibiting only a part of the continuous amino acid sequence orsecondary conformations within TIMP-3 possessing the ability to bind tothe extracellular matrix are also specifically contemplated herein.

It is noteworthy that activity is not necessary for any one or more ofthe products of the invention to have therapeutic utility (see, Weilandet al., Blut 44: 173–175 (1982) or utility in other contexts, such as inassays of TIMP-3 antagonism. Competitive antagonists may be quite usefulin, for example, cases of overproduction of TIMP-3.

Of applicability to TIMP-3 fragments and polypeptide analogs of theinvention are reports of the immunological activity of syntheticpeptides which substantially duplicate the amino acid sequence extant innaturally-occurring proteins, glycoproteins and nucleoproteins. Morespecifically, relatively low molecular weight polypeptides have beenshown to participate in immune reactions which are similar in durationand extent to the immune reactions of physiologically significantproteins such as viral antigens, polypeptide hormones, and the like.Included among the immune reactions of such polypeptides is theprovocation of the formation of specific antibodies in immunologicallyactive animals. See, e.g., Lerner et al., Cell 23: 309–310 (1981); Rosset al., Nature 294: 654–656-(1981); Walter et al., PNAS-USA 77:5197–5200 (1980); Lerner et al., PNAS-USA, 78: 3403–3407 (1981); Walteret al., PNAS-USA 78: 4882–4886 (1981); Wong et al., PNAS-USA 79:5322–5326 (1982); Baron et al., Cell 28: 395–404 (1982); Dressman etal., Nature 295: 185–160 (1982); and Lerner, Scientific American 248:66–74 (1983). See, also, Kaiser et al. Science 223: 249–255 (1984)relating to biological and immunological activities of syntheticpeptides which approximately share secondary structures of peptidehormones but may not share their primary structural conformation.

One type of analog is a truncated TIMP-3 having capacity to bind to thezinc binding domain of collagenase. For example, the zinc binding domainon interstitial collagenase is located at amino acids 218, 222 and 228at the pro enzyme. Goldberg, G. I., J. Biol. Chem. 261: 6600–6605(1986). The zinc binding domain of the 72 kDa species of procollagenaseis located at amino acids 403–407. Collier et al., Genomics 9: 429–434(1991). The zinc binding domain of the 92 kDa species of procollagenaseis at amino acids 401–405. Van Ranst et al., Cytokine 3: 231–239 (1991).Interestingly, the zinc binding domain is fairly well conserved amongenzymes: H E F G H (SEQ ID NO:37, 92 kDa collagenase), H E F G H (SEQ IDNO:37, 72 kDa collagenase) and H E L G H (SEQ ID NO:38, interstitialcollagenase). Thus, the motif for zinc binding is H E X G H (SEQ IDNO:42) wherein X is either F or L. A selective binding molecule, such asan antibody or small molecule would block such zinc binding andtherefore inhibit enzymatic activity. (The term “selective bindingmolecule” as used here indicating a composition which selectively bindsto its target.) One may prepare a monoclonal antibody or a recombinantantibody, for example.

TIMP-2 deletion analogs have been prepared which have retained theability to inhibit metalloproteinase activity, Willenbrock et al.,Biochemistry 32: 4330–4337 (1993). For TIMP-2, the C-terminus wasshortened to delete six C-terminal cysteines (three disulfide-bondedloops). Thus, in view of the homology among the various zinc bindingdomains, one could prepare analogous TIMP-3 analogs with similarlyshortened C-terminal sequences. The TIMP-3 analog 1–121 (using thenumbering of FIG. 1 herein) includes the first six cysteines residues,but not the last six. One may optionally lengthen the C-terminus up tothe full length molecule of 188 amino acids. Such analogs may also beprepared for any species, such as ChIMP-3.

This is further demonstrated below in the examples, as a TIMP-2 deletionvariant is shown to inhibit interstitial collagenase. (Example 3 below).The zinc binding domain of interstitial collagenase is similarlysituated as that of the 72 kDa species collagenase (which was shown byWillenbrock et al., supra, to be affected by the TIMP-2 truncatedanalogs).

Also, since it is apparent that the C-terminus is not necessary forenzyme inhibition activity, one may chemically modify the C-terminus.One may desire, for example, a sustained release preparation whereby oneor more polymer molecules such as polyethylene glycol molecules areattached. Other chemical modifications include attachment of anadditional polypeptide for the creation of a fusion molecule. Thus,another aspect of the present invention is chemically modified TIMP-3.

The present invention also includes that class of polypeptides coded forby portions of the DNA complementary to the protein-coding strand of thehuman cDNA or genomic DNA sequences of TIMP-3 i.e., “complementaryinverted proteins” as described by Tramontano et al. Nucleic Acid Res.12: 5049–5059 (1984). Polypeptides or analogs thereof may also containone or more amino acid analogs, such as peptidomimetics.

Also comprehended by the invention are pharmaceutical compositionscomprising effective amounts of polypeptide products of the inventiontogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers useful in TIMP-3therapy. Such compositions include diluents of various buffer content(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additivessuch as detergents and solubilizing agents (e.g., Tween 80, Polysorbate80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances(e.g., lactose, mannitol); covalent attachment of polymers such aspolyethylene glycol to the protein (as discussed supra, see, for exampleU.S. Pat. No. 4,179,337 hereby incorporated by reference); incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes. Suchcompositions will influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of TIMP-3. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435–1712 which are herein incorporated byreference.

Generally, an effective amount of the present TIMP-3 polypeptides willbe determined by the age, weight and condition or severity of disease ofthe recipient. See, Remingtons Pharmaceutical Sciences, supra, at pages697–773, herein incorporated by reference. Typically, a dosage ofbetween about 0.001 g/kg body weight to about 1 g/kg body weight, may beused, but more or less, as a skilled practitioner will recognize, may beused. For local (i.e., non-systemic) applications, such as topicalapplications, the dosing may be between about 0.001 g/cm² to about 1g/cm². Dosing may be one or more times daily, or less frequently, andmay be in conjunction with other compositions as described herein. Itshould be noted that the present invention is not limited to the dosagesrecited herein.

A plurality of agents act in concert in order to maintain the dynamicequilibrium of the extracellular matrix and tissues. In treatment ofconditions where the equilibrium is skewed, one or more of the otheragents may be used in conjunction with the present TIMP-3. These otheragents may be co-administered or administered in seriatim, or acombination thereof. Generally, these other agents may be selected fromthe list consisting of the metalloproteinases, serine proteases,inhibitors of matrix degrading enzymes, intracellular enzymes, celladhesion modulators, and factors regulating the expression ofextracellular matrix degrading proteinases and their inhibitors. Whilespecific examples are listed below, one skilled in the art willrecognize other agents performing equivalent functions, includingadditional agents, or other forms of the listed agents (such as thoseproduced synthetically, via recombinant DNA techniques, and analogs andderivatives).

Metalloproteinases and serine proteases degrade the extracellularmatrix, as discussed above. Thus, use of enzymes in therapy may be tocounteract effects of the present TIMP-3, which inhibits suchdegradation. Enzymes include collagenases, PMN (polymorphonuclearleukocyte) collagenase, stromelysin I, II/transin, matrilysin,invadolysin, putative metalloproteinase (PUMP-1), urokinase typeplasminogen activator (UPA), tissue plasminogen activator (TPA), andplasmin. PD-ECGF may also be used.

Other degradation inhibitors may also be used if increased or morespecific prevention of extracellular matrix degradation is desired.Inhibitors may be selected from the group consisting of α₂macroglobulin, pregnancy zone protein, ovostatin,. α₁-proteinaseinhibitor, α₂-antiplasmin, aprotinin, protease nexin-1, plasminogenactivator inhibitor (PAI)-1, PAI-2, TIMP-1, and TIMP-2. Others may beused, as one skilled in the art will recognize.

Intracellular enzymes may also be used in conjunction with the presentTIMP-3. Intracellular enzymes also may affect extracellular matrixdegradation, and include lysozomal enzymes, glycosidases and cathepsins.

Cell adhesion modulators may also be used in combination with thepresent TIMP-3. For example, one may wish to modulate cell adhesion tothe extracellular matrix prior to, during, or after inhibition ofdegradation of the extracellular matrix using the present TIMP-3. Cellswhich have exhibited cell adhesion to the extracellular matrix includeosteoclasts, macrophages, neutrophils, eosinophils, killer T cells andmast cells. Cell adhesion modulators include peptides containing an“RGD” motif or analog or mimetic antagonists or agonists.

Factors regulating expression of extracellular matrix degradingproteinases and their inhibitors include cytokines, such as IL-1 andTNF-α, TGF-β, glucocorticoids, and retinoids. Other growth factorseffecting cell proliferation and/or differentiation may also be used ifthe desired effect is to inhibit degradation of the extracellular matrixusing the present TIMP-3, in conjunction with such cellular effects. Forexample, during inflammation, one may desire the maintenance of theextracellular matrix (via inhibition of enzymatic activity) yet desirethe production of neutrophils; therefore one may administer G-CSF. Otherfactors include erythropoietin, interleukin family members, SCF, M-CSF,IGF-I, IGF-II, EGF, FGF family members such as KGF, PDGF, and others.One may wish additionally the activity of interferons, such asinterferon alpha's, beta's, gamma's, or consensus interferon.Intracellular agents include G-proteins, protein kinase C and inositolphosphatases. While the field of inflammation research is presentlyunder development, and the precise interactions of the describedcompositions in vivo is not throughly understood, the use of the presentpolypeptides may provide therapeutic benefit with one or more agentsinvolved in inflammation therapy.

Cell trafficking agents may also be used. For example, inflammationinvolves the degradation of the extracellular matrix, and the movement,or trafficking of cells to the site of injury. Prevention of degradationof the extracellular matrix may prevent such cell trafficking. Use ofthe present TIMP-3 in conjunction with agonists or antagonists of celltrafficking-modulation agents may therefore be desired in treatinginflammation. Cell trafficking-modulating agents may be selected fromthe list consisting of endothelial cell surface receptors (such asE-selectins and integrins); leukocyte cell surface receptors(L-selectins); chemokins and chemoattractants. For a review ofcompositions involved in inflammation, see Carlos et al., Immunol. Rev.114: 5–28 (1990), which is herein incorporated by reference.

Moreover, compositions may include neu differentiation factor, “NDF,”and methods of treatment may include the administration of NDF before,simultaneously with, or after the administration of TIMP-3. NDF has beenfound to stimulate the production of TIMP-2, and the combination of NDF,TIMP-1, -2 and/or -3 may provide benefits in treating tumors.

Polypeptide products of the invention may be “labeled” by associationwith a detectable marker substance (e.g., radiolabeled with ¹²⁵I) toprovide reagents useful in detection and quantification of TIMP-3 insolid tissue and fluid samples such as blood or urine. Nucleic acidproducts of the invention may also be labeled with detectable markers(such as radiolabels and non-isotopic labels such as biotin) andemployed in hybridization processes to locate the human TIMP-3 geneposition and/or the position of any related gene family in a chromosomalmap. Nucleic acid sequences which selectively bind the human TIMP-3 geneare useful for this purpose. They may also be used for identifying humanTIMP-3 gene disorders at the DNA level and used as gene markers foridentifying neighboring genes and their disorders. Contemplated hereinare kits containing such labelled materials.

The TIMP-3 compositions described herein modify the pathogenesis andprovide a beneficial therapy for diseases of connective tissuescharacterized by matrix degradation. Also, the present TIMP-3compositions may be useful in the treatment of any disorder whereexcessive matrix loss is caused by metalloproteinase activity. TheTIMP-3 compositions may be used alone or in conjunction with one or moreof the agents discussed herein.

Polypeptide products of the present invention are useful, alone or incombination with other drugs, in the treatment of various disorders suchas dystrophic epidermolysis bullosa where the disease is linked to theoverproduction of collagenase, Bauer et al., J. Exp. Med. 148: 1378–1387(1978). The products of the present invention may also be useful in thetreatment of rheumatoid arthritis. Evanson et al. J. Clin. Invest. 47:2639–2651 (1968) noted that large amounts of collagenase are produced,in culture, by excised rheumatoid synovial tissue, this led toimmunolocalization studies by Woolley et al., Arthritis and Rheumatism20: 1231–1239 (1977), with monospecific antibodies directed againsthuman rheumatoid synovial collagenase which detected high levels ofimmunoreactive collagenase at the sites of joint erosion(cartilagepannus junctions) but not in the cartilage of associatedchondrocytes, and not in the synovium at sites remote from the resorbingfront. Collagenases have also been demonstrated using many otherdifferent preparations derived from human rheumatoid joints and usingtissues characterized by other types of arthritis such asosteoarthritis, Reiter's syndrome, pseudogout, juvenile rheumatoidarthritis, and scleroderma.

In periodontal disease affecting the tooth supporting apparatus,elevation of collagenolytic enzymes is evident, and destruction ofcollagen and connective tissue. See, V.-J. Uitto, pp. 211–223 inProteinases in Inflammation and Tumor Invasion, H. Tschesche, ed.,Walter de Gruyter & Co., Berlin, N.Y. (1988).

Collagenases have been implicated in ulceration including corneal,epidermal, or gastric ulceration, Brown et al., American J. ofOphthalmology 72: 1139–1142 (1971), and, indeed, metalloproteinaseinhibitors are used in the treatment of corneal ulceration. Slansky etal., Annals of Ophthalmology 2: 488–491 (1970).

In wound healing after surgery, TIMP-3 may have particular applicationfor restenosis. Metalloproteinases contribute to the rearrangement ofarterial cells, including blockage of the artery. Use of the presentTIMP-3 may inhibit such arterial wall rearrangement. Delivery ofantisense TIMP-3 nucleic acid may also provide benefit.

In the field of tumor invasion and metastasis, the metastatic potentialof some particular tumors correlates with the increased ability tosynthesize and secrete collagenases, Liotta et al., Nature 284: 67–68(1980), and with the inability to synthesize and secrete significantamounts of a metalloproteinase inhibitor, Hicks et al., Int. J. Cancer33: 835–844 (1984). These processes are related to the passage of tumorcells through connective tissue layers (basement membrane) from tissuesites to the circulation and vice versa, which could be retarded byTIMP-3. TIMP-3 similarly has therapeutic application in inhibiting tumorcell dissemination during removal of primary tumors, during chemotherapyand radiation therapy, during harvesting of contaminated bone marrow,and during shunting of carcinomatous ascites.

A limiting factor in the use of bone marrow transplantation for manyadvanced cancers with bone marrow involvement is the absence of adequatepurging techniques. For example, metastatic interstitial pneumonitisfollowing infusion of improperly purged bone marrow cells has beennoted, Glorieux et al., Cancer 58: 2136–2139 (1986); Graeve et al.,Cancer 62: 2125–2127 (1988). TIMP-3 administered during infusion ofunpurged bone marrow cells will alleviate the need for developingexpensive purging techniques.

Diagnostically, correlation between absence of TIMP-3 production in atumor specimen and its metastatic potential is useful as a prognosticindicator as well as an indicator for possible prevention therapy.

Tumors may also become more or less encapsulated or fibrotic due toincreased collagen deposition (or inhibition of breakdown) by bothcancer cells and/or surrounding normal cells. Increased encapsulationpromoted by TIMP-3 aids in clean tumor excision.

Other pathological conditions in which excessive collagen degradationmay play a role and thus where TIMP-3 can be applied, include emphysema,Paget's disease of bone, osteoporosis, scleroderma, pressure atrophy ofbone or tissues as in bedsores, cholesteatoma, and abnormal woundhealing. TIMP-3 can additionally be applied as an adjunct to other woundhealing promoters, e.g., to modulate the turnover of collagen during thehealing process.

TIMP-3 also may have erythroid potentiating activity (i.e., stimulationof differentiation of early erythroid progenitors), and thus TIMP-3 maybe useful in the treatment of various anemias.

In addition TIMP-3 may have application in the treatment ofimmunological disorders such as autoimmune diseases (e.g., rheumatoidarthritis, multiple sclerosis) based upon a potential ability tosuppress B-cell differentiation as determined by the method of Pisko etal., J. Immunol. 136: 2141–2150 (1986).

Based on its ability to inhibit connective tissue degradation, TIMP-3and/or other TIMP molecules have application in cases where inhibitionof angiogenesis is useful, e.g., in preventing or retarding tumordevelopment, and the prevention of the invasion of parasites. Inaddition, the present compositions and methods may be applicable forcosmetic purposes, in that localized inhibition of connective tissuebreakdown may alter the appearance of tissue.

The present compositions and methods may also be useful in birth controlor fertilization modulation as the TIMPs have been shown to prevent orretard follicular rupture, Branstrom et al., Endocrinology 122:1715–1721 (1988), and interfere with embryo preimplantation development.

The present compositions and methods may be useful in the treatment ofnerve cell disorders in that TIMP-3 may protect nerve cells from damageby preserving the basement membrane surrounding nerve cells. Therefore,uses may involve BDNF, NT-3, NGF, CNTF, NDF, SCF, or other nerve cellgrowth or proliferation modulation factors.

As described above, the present TIMP-3 has wide application in a varietyof disorders. Thus, another embodiment contemplated herein is a kitincluding the present polypeptides and optionally one or more of theadditional compositions described above for the treatment of a disorderinvolving the degradation of extracellular matrix. An additionalembodiment is an article of manufacture comprising a packaging materialand a pharmaceutical agent within said packaging material, wherein saidpharmaceutical agent contains the present polypeptide(s) and whereinsaid packaging material comprises a label which indicates that saidpharmaceutical agent may be used for an indication selected from thegroup consisting of: cancer, inflammation, arthritis, dystrophicepidermolysis bullosa, periodontal disease, ulceration, emphysema, bonedisorders, scleroderma, wound healing, erythrocyte deficiencies,cosmetic tissue reconstruction, fertilization or embryo implantmodulation, and nerve cell disorders. This article of manufacture mayoptionally include other compositions or label descriptions of othercompositions.

The nucleic acids provided herein may also be embodied as part of a kitor article of manufacture. Contemplated is an article of manufacturecomprising a packaging material and a pharmaceutical agent, wherein saidpharmaceutical agent contains the presently provided nucleic acids andwherein said packaging material comprises a label which indicates thatsaid pharmaceutical composition may be used for an indication benefitingfrom the modulation of said DNA expression, such as a gene therapyindication. Such gene therapy indications, as discussed above, includethe treatment of emphysema. A kit containing the nucleic acid(s) mayinclude, optionally, additional factors affecting the ex vivo growth ofrecipient-cells, such as SCF. See, e.g., Zsebo et al., PCT WO 91/05795,herein incorporated by reference.

A further embodiment of the invention is selective binding molecules,such as monoclonal antibodies specifically binding TIMP-3. The hybridomatechnique described originally by Kohler and Milstein Eur. J. Immunol.6, 511–519 (1976) has been widely applied to produce hybrid cell linesthat secrete high levels of monoclonal antibodies against many specificantigens. Recombinant antibodies, (see Huse et al., Science 246: 1275(1989)) may also be prepared. Such antibodies may be incorporated into akit for diagnostic purposes, for example.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1

Cloning and Expression of Human TIMP-3 cDNA

The overall cloning strategy involved two steps, the first, obtaining afragment using PCR from a human fetal kidney cDNA library, and thesecond, using this partial clone to screen two different cDNA librariesfor full length cDNA sequences.

Degenerate PCR primers derived from highly conserved regions of the TIMPgene family were used to amplify TIMP-3 cDNA from human fetal kidneycDNA. This product was then used as a probe to isolate clones from ahuman fetal kidney cDNA library and a normal human colonic mucosa cDNAlibrary. Clones of 1240, 963 and 827 bp have been isolated andsequenced. The longest clone encodes the entire 211 amino acidpro-polypeptide, having a mature polypeptide of 188 amino acids. Theintermediate size clone is truncated but encodes the entire matureprotein. The smallest clone is missing the region encoding the first 24amino acids of the mature polypeptide. Also demonstrated is theexpression and purification of mature polypeptide.

Materials and Methods

Primers and Initial TIMP-3 DNA Source Used.

Degenerate PCR primers were used in a first round screening of firststrand cDNAs to obtain a partial TIMP-3 cDNA clone. The degenerate PCRprimers were derived from highly conserved regions of the TIMP family ofproteins were selected, (see FIG. 4). They were also chosen because ofthe relatively low degeneracy of their codons.

The forward primer was derived from a sequence (VIRA, SEQ ID NO:39)which is ubiquitous throughout the TIMP family and is found at positions18–21 of the mature proteins. This 96-fold degenerate forward primer had11 bases that encoded the TIMP sequence plus 6 bases for an EcoRI siteand 2 extra bases (underlined) 449-15: SEQ. ID No. 1: 5′-CGG AAT TCG TNATHM GNG C-3′

A reverse primer corresponding to a region of ChIMP-3 (CIWTDM, SEQ IDNO:40) was synthesized. This primer, 480-27, included a BamHI site andtwo extra bases (underlined): SEQ. ID No. 2 5′-CGG GAT CCC ATR TCN GTCCAD ATR CA-3′.

An alternative reverse primer was also used: SEQ. ID No. 3 480-28 CGGGAT CCR TCN GTC CAD ATR CA

The corresponding region is somewhat variant. Amino acids 163–168 ofChIMP-3 are encoded by the version used here, and these were chosenbecause the M and I distinguished the ChIMP-3 from other TIMPs. It wasnot initially known if these differences would also be present in humanTIMP-3 (if such TIMP did indeed exist), however, a bias away from TIMP-1and TIMP-2 was used to limit unwanted amplifications. The M at position168 was especially useful. As a result of its location at the 5′ end ofthe reverse primer, it would not interfere with the PCR process if therewere mismatches and it would favor TIMP-3 amplification over other DNAsif this choice were correct.Amplification of First Strand cDNAs Using Primers

First, the degenerate primers were used to amplify PCR products from thetwo first strand cDNAs. After a second round of amplification the PCRproducts of these were subcloned, and one was selected which was used asa probe for cDNA libraries, as described below.

Oligonucleotide synthesis. Oligonucleotides were synthesized on AppliedBiosystems 394 automated synthesizers using standard phosphoramiditechemistry. Degenerate oligonucleotides, which were synthesized ingreater than 200 nmole quantities, were purified by butanol extraction.Nondegenerate oligonucleotide were synthesized in smaller amounts andwere purified Trityl-on using Poly-pak (Glen Research Corp., Sterling,Va.) cartridges following the manufacturer's instructions. Trityl-offpurification was done using 1×25 cm Sephadex G-50 chromatography columnsand TEAB as the elution buffer.

Polymerase Chain Reaction. All PCR was performed on Perkin Elmer model9600 instruments using Perkin Elmer Cetus (Norwalk, Conn.) GeneAmp kitsaccording to the manufacturer's instructions which are hereinincorporated by reference.

The first round of PCR consisted of 5 cycles at 94° C. for 20 seconds,50° C. for 20 seconds and 72° C. for 30 seconds. This was followed by 30cycles at 94° C. for 20 seconds, 50° C. for 20 seconds and 72° C. for 30seconds. The PCR products were run on a 2% agarose (SeaKem GTG, FMC,Rockland, Me.) gel, prestained with ethidium bromide (Sigma, St. Louis,Mo.), and the bands in the predicted size range were punched out of thegel using a Pasteur pipette. The PCR products were then re-amplifieddirectly from the gel fragments using the same PCR primers and thefollowing program: 1 cycle of 5 minutes at 95° C. followed by 25 cyclesof 94° C. for 20 seconds, 50° C. for 20 seconds, and 72° C. for 30seconds. This process was performed a second time in an effort to obtainlarge quantities of relatively pure material for subcloning andrestriction analysis.

First Strand cDNA Sources Oligo dT-primed first strand cDNA from humancolonic mucosa (Dr. Gene Finley, Pittsburgh VA Medical Center) as welloligo dT-primed first strand cDNA from 22 week old human fetal kidney(Clontech, Palo Alto, Calif.) were used as first-round sources of TIMP-3cDNA. When the colonic mucosa cDNA source was used, the same bandingpattern was observed as that observed with the fetal kidney cDNAs, whichconfirmed those results. These fetal kidney PCR products were then usedfor subcloning.

Purification and Subcloning of PCR Products. The PCR products were runthrough Centricon-100 columns (Amicon, Beverly, Mass.) to facilitate theDNA to be cleaved with restriction endonucleases. The DNA was then cutwith EcoRI and BamHI to ensure that they would not be internally cleavedduring the subcloning process. PCR products were cloned into pUC19 aftertreatment with proteinase K (Crowe et al., 1991) to enhance the cloningefficiency. Colonies were rapidly screened by PCR amplification withvector primers 382-3 SEQ. ID No. 4 (5′-GTT TTC CCA GTC ACG ACG-3′) and382-4 SEQ. ID No. 5 (5′-GAA TTG TGA GCG GAT AAC-3′). These products werepurified using Centricon-100 concentrators and were sequenced.

Results. As shown in FIG. 2 three bands resulted from amplification withthe degenerate primers. Cloned DNA from two of the bands was sequenced;the third band could not be purified sufficiently to allow subcloningand sequencing.

The smaller of the two sequenced bands was the desired 402 bp fragmentand the larger band presumably resulted from false priming to the regionencoding CSWYRG (amino acids 169–174 of the mature polypeptide ofFIG. 1) and was 489 bp. The 402 bp fragment corresponds to the nucleicacid encoding the region encompassing ValIleArgAla(Lys) toCysLeuTrpThrAspMet of FIG. 1, with an EcoRI on the 5′ side, and an BamHIon the 3′ side. Also, the codon for isoleucine on the 3′ end is replacedwith the codon for leucine.

cDNA Library Screening

Screening of a First cDNA Library.

Library. The first library screened was an the oligo(dT)-primed λgt11Clontech human 20 and 24 week fetal kidney cDNA library (Clontech).

Probes. The first round of cDNA screening was done with the insert ofone of the cloned degenerate PCR products previously described, the 402bp insert. A low level of background was observed as a result ofcontamination with pUC19 vector DNA. Consequently, the phage supernatantfrom a partially purified λgt11 clone obtained from the first round ofcDNA screening was used as a PCR template. Friedman et al., Nucl. AcidsRes. 17: 8718 (1988). This provided a probe of high quality and purity.The Primer 495-21, SEQ. ID No. 6 5′-CGG AAT TCT GGT CTA CAC CAT CAAGC-3′ corresponded approximately to the YTIK domain and including anEcoRI site and two additional bases. Primer 496–16, SEQ. ID No. 7 5′-CATGTC GGT CCA GAG ACA CTC G-3′, corresponded to the CLWTDM region and didnot include any restriction sites. This resulted in a 333 bp fragment.The sequence of the 333 bp fragment was a portion of the 402 bp fragmentsequence. The 333 bp fragment was used as a probe for all of thenorthern blot analyses and for all further cDNA library screening. The333 bp fragment corresponds to the region of FIG. 1 encodingTyrThrIleLys through CysLeuTrpThrAspMet and the EcoRI site mentionedabove.

Plaque Hybridization About 200,000 phage were plated on ten 150 mmplates, lifted in duplicate onto Schleicher & Schuell supportednitrocellulose membranes and probed with a randomly primed, ³²P-labeled(Stratagene) 402 bp fragment described above. Prehybridizations andhybridizations were performed overnight at 42° C. using the followingreagents (for 50 ml of solution):

12.5 ml 20X SSPE 5 ml −0.5 N NaHPO₄ pH 6.8 0.1 ml 0.50 M EDTA pH 8.0 25ml formamide 2.5 ml 50X Denhardt's 0.25 ml 20% SDS 0.5 ml 10 mg/ml tRNA(calf's liver) 1 ml 10 mg/ml salmon sperm DNA (not used in thepre-hybridization solution) 4.15 ml H₂O (3.15 ml used in thehybridization solution)

The filters were washed in 0.25×SSC at 42° C. Two positively hybridizingplaques were purified, resulting in 2 independent clones here namedTimp3clone7 and Timp3clone2. DNA from bacteriophage lambda was purifiedusing a Qiagen Lambda DNA purification kit (Chatsworth, Calif.). Platelystates from 10 confluent 135 mm petri dishes were pooled for eachspecimen. 300 μl of a solution containing 20 mg/ml RNase, 6 mg/ml DNaseI, 0.2 mg/ml BSA, 10 mM EDTA, 100 mM Tris-HCl, 300 mM NaCl, pH 7.5 wereadded and incubated at 37° C. for 30 minutes. 10 ml of ice cold 30%polyethylene glycol (PEG 6000), 3 M NaCl were mixed in and incubated onice for 60 minutes.

After centrifugation at 10,000×g for 10 minutes, the supernatant wasdiscarded. The pellet was resuspended in 10 ml of a solution containing100 mM Tris-HCl, 100 mM NaCl and 25 mM EDTA, pH 7.5. 10 ml of a solutioncontaining 4% SDS was gently added and the mixture was heated at 70° C.for 10 minutes and then cooled on ice. 10 ml of 2.55 M potassiumacetate, pH 4.8 was mixed in quickly and the solution was centrifuged at4° C. at 15,000×g for 30 minutes. The supernatant was run on a Qiagentip-500 column which had been equilibrated with 10 ml of 750 mM NaCl, 50mM MOPS, 15% ethanol, pH 7.0. The column was then washed with 30 ml 1.0M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0. Finally, the column was elutedwith 15 ml of 1.25 M NaCl, 50 mM MOPS, 15% ethanol, pH 8.2. The eluatewas precipitated in 0.7 volumes of isopropanol and centrifuged at 4° C.for 30 minutes. The pellet was air dried for 5 minutes and cut withBoehringer Mannheim (Mannheim, Germany) high concentration EcoRI.

The inserts which had hybridized to the 333 bp probe were purified fromagarose gel slices using a Qiaex DNA extraction kit (Qiagen, Chatsworth,Calif.). A solution of 3 M NaI, 4 M NaClO₄, 5 mM Tris-H, pH 7.5 at threetimes the volume of the gel slice was added, along with 0.1 times thegel slice volume of 1 M mannitol and 10 ml of Qiaex resin in a 1.5 mlmicrocentrifuge tube. This mixture was heated at 50° C. for 10 minutesor until the agarose is completely dissolved. The DNA was allowed toadsorb at room temperature for 5 minutes and then the tubes were brieflycentrifuged (6 seconds). After the supernatants were discarded, theQiaex resin in the tubes were washed in a solution containing 8 MNaClO₄, and centrifuged (6 seconds). This wash and centrifugation wasrepeated and was followed by 2 washes (each followed by 6-secondcentrifugations) in a solution containing 70% ethanol, 100 mM NaCl, 10mM Tris-HCl, 1 mM EDTA, pH 7.5. The resin was air dried and eluted in 20μl of water.

The purified inserts were cloned into pUC19 (New England Biolabs) usingBoeringer Mannheim's T4 DNA polymerase. There was an insert to vector(molar) ratio of approximately 5:1. Ligations were performed overnightat 14° C. The ligated material was ethanol precipitated in the presenceof glycogen to increase the recovery. This material was thenelectroporated into BRL's (Gibco-BRL, Gathersburg, Md.) electroporationcompetent DH10B cells.

Preparations of plasmid DNA were made using using Qiagen plasmid DNApurification kit. A. 10 ml overnight culture of a single bacterialcolony was grown in terrific broth [Tartoff and Hobbs, Bethesda Res.Lab. Focus 9:12 (1987). Per liter: 12 g bacto-tryptone, 24 g bacto-yeastextract, 4 ml glycerol] with 50 μg/ml ampicillin. The overnight growthwas used to inoculate a 250 ml culture in a sterile 1-liter baffledflask containing terrific broth with 50 μg/ml ampicillin. After thisgrew to saturation, the medium was centrifuged at 5000 rpm for 10minutes. The bacterial pellet was resuspended in 10 ml of 100 μg/mlRNaseA, 50 mM Tris-HCl. 10 ml of 200 mM NaOH, 1% SDS was added to theresuspended pellet and the mixture was incubated at room temperature for5 minutes. 10 ml of 2.55 M KAc, pH 4.8 was added and mixed gently. Thematerial was immediately centrifuged at 10000 rpm for 10 minutes. Thesupernatant was filtered through a cotton gauze pad and the lysate thatwas particle-free was added to a Qiagen tip-500 column following thesame procedure as per the lambda DNA preparation procedure.

Screening of a second cDNA library. A cDNA library from human colonicmucosa, kindly provided by Jim Pipas of the University of Pittsburgh,was the second library screened for TIMP-3 cDNA. This library usedUni-Zap (Stratagene, La Jolla, Calif.) as the vector and had a titer of2.4×10¹⁰ pfu/ml. Hybridization was performed as with the kidney library,using the 333 bp probe. The Uni-Zap vector has a pBluescript phagemidwhich was excised from the phage to which the probes hybridized, andsequenced directly.

Phage particles were isolated and amplified as follows. Phage particleswere released into the SM buffer by incubating for 2 hours at roomtemperature. In a 50 ml test tube, 200 μl of O.D.₆₀₀=1.0 XL1-Blue cellsand 200 μl of the lambda Zap phage were combined with 1 ml of R408helper phage which had a titer of 10¹⁰ pfu/ml. This mixture wasincubated at 37° C. for 15 minutes. 3 ml of 2×YT medium (per liter: 16 gbacto-tryptone, 10 g bacto-yeast extract, 5 g NaCl) were added and themixture was then incubated for 2.5 hours at 37° C. with shaking. Thetube was heated at 70° C. for 20 minutes and then centrifuged at 4000×gfor 5 minutes.

To rescue the phagemid, 50 μl of the heat-disrupted phage stock wereincubated with 200 μl of O.D.₆₀₀=1.0 XL1-Blue cells in a 1.5 ml tube.Additionally, 10 μl of a 10⁻² dilution of heat-disrupted phage wereincubated with 200 μl of O.D.₆₀₀=1.0 XL1-Blue cells in a separate 1.5 mltube. The tubes were incubated at 37° C. for 15 minutes and the cellswere then plated on LB ampicillin plates and incubated overnight at 37°C. Colonies appearing on the plate contained the pBluescript SK-doublestranded phagemid with the cloned DNA insert.

This screening resulted in one clone, here named “TIMP3HCM3,” (see FIG.16), lacking a portion encoding the N-terminus of the maturepolypeptide.

DNA Sequencing

All sequencing was performed on Applied Biosystems, Inc. (ABI) 373AAutomated Sequencers. PCR products were sequenced using nested pUCvector dye-primers and ABI's catalyst to perform the reactions.

Double stranded cDNAs cloned into pUC19 were sequenced using ABI's PrismReady Reaction Dye-Deoxy Terminator Cycle Sequencing Kit using theprotocol that came with the kit. For areas of high GC content leading tohairpin loops, reactions were done with the following changes from thestandard kit protocol: denaturation at 98° C. for 30 seconds, 12 UAmplitaq, substitution of New England Biolabs (NEB) Vent Polymerasebuffer for the ABI TACS buffer and, 30 cycles instead of 25 cycles.

Sequence Analysis

DNA and deduced amino acid analyses used the Genetics Computer Group(GCG) sequence analysis software package from the University ofWisconsin Department of Genetics, Genetic Computer Group, Inc.,University Research Park, 575 Science Drive, Suite B, Madison, Wis.53711.

Expression of Recombinant Human TIMP-3 in E. coli

The coding sequence of Timp3clone7 (ATCC Accession No. 69454) wasamplified by PCR using standard kit protocol. All deposits were madewith the American Type Culture Collection, P.O. Box 1549, Manassas, Va.20108, USA. Primer 544-29 SEQ. ID No. 8 (5′-AAC AAA CAT ATGTGC ACA TGCTCG CCC AGC C-3′) consists of nucleotides 351 to 369, which encodesTIMP-3 amino acids 24–29 (1–6 of the mature protein of FIG. 1). An NdeIsite and 6 extra bases (underlined) were included to facilitatesubcloning into a bacterial expression vector. The methionine initiatorcodon, (italics), was added to facilitate expression. The downstreamprimer, 532-13, SEQ. ID No. 9 (5′-CGG GAT CCT ATT AGG GGT CTG TGG CATTGA TG-3′) corresponds to nucleotides 896 to 914 (of FIG. 1) with anadded BamHI site and 2 additional bases (underlined) as well as two stopcodons (italicized). The naturally occurring stop codon, TGA (TCA on thereverse complement) was changed to TAA (TTA on the reverse complement),since it is a more efficient stop in E. coli. The second stop codon. TG,(CTA on the reverse complement) was added as a backup.

The vector pCFM3102, as described below, was digested with NdeI andBamHI overnight as was the 589 bp PCR fragment encoding TIMP-3. Thereaction was stopped by extraction with phenol/chloroform followed byextraction with chloroform alone. The aqueous layer was then passedthrough a 1 ml Sephadex G-50 spin column (in a 1 ml syringe) that wasequilibrated with 200 μl 10 mM Tris-HCl, 1 mM EDTA pH 8.0. Theflow-through from the column was collected and precipitated with 0.1volumes of 3 M NaAc, pH 5.4 and 2.5 volumes of 100% ethanol. Aftercentrifugation, the pellet was washed in 70% ethanol and dried in aSpeed-Vac (Savant). The pellets were resuspended in 20 μl Super-Q water.

A mock ligation containing cut pCFM3102 with no insert was done inaddition the TIMP-3::pCFM3102 ligation. Ligations were performedovernight at 14° C., using Boehringer Mannheim T4 DNA ligase. They werethen precipitated, washed and dried as above. The pellets were thenresuspended in 5 μl of Super-Q water. 2.5 μl of each ligation was usedto electroporate 40 μl of electroporation competent cells.

Electroporation of plasmid into E. coli occurred in 0.1 cm cuvettes(Bio-Rad) at 1.9 kV, 200 ohms, 25 μF using a Bio-Rad Gen Pulser and withimmediate recovery in 5 ml of SOC medium. The cells recovered at 28° C.for 11.3 hours and were plated out onto LB plates containing kanamycin.The plates were incubated at 28° C. overnight. Colonies were screenedfor inserts by PCR using vector-specific primers 315-21 SEQ. ID No. 10(5′-ACC ACT GGC GGT GAT ACT GAG-3′) and 315-22 SEQ. ID No. 11 (5′-GGTCAT TAC TGG ACC GGA TC-3′). Colonies having inserts gave PCR productsthat are 589 bp larger than the PCR product derived from the originalvector without an insert.

Construction of Expression Plasmid pCFM3102

Expression of the mature protein was accomplished in E. coli using aplasmid vector. A culture of this E. coli, containing plasmid encoding amature polypeptide as presented in FIG. 1, is deposited at the ATCC,accession no. 69455.

The plasmid used was derived from pCFM836, which is fully described inU.S. Pat. No. 4,710,473, herein incorporated by reference. Theconstruction for the present plasmid (denominated pCFM3102) from thedescribed pCFM836 plasmid (U.S. Pat. No. 4,710,473) was by destroyingthe two endogenous NdeI restriction sites, by end filling with T4polymerase enzyme followed by blunt end ligation, by replacing the DNAsequence between the unique AatII and ClaI restriction sites containingthe synthetic P_(L) promoter with a similar fragment obtained frompCFM636 (U.S. Pat. No. 4,710,473) containing the P_(L) promoter, bysubstituting the small DNA sequence between the unique ClaI and KpnIrestriction sites with an oligonucleotide containing a number ofrestriction sites, and by making a series of site directed base changesby PCR overlapping oligonucleotide mutagenesis through the intermediatepCFM1656 vector (4799 base pair).

Fermentation

The inoculum for the fermentation was started by transferring 0.1 ml ofa glycerol stock at 1 O.D./ml in LB+17% glycerol of ATCC Accession No.69455 (E. coli host cells containing the pCFM3102 with inserted TIMP-3coding sequences) into a 2-L nippled flask containing 500 ml of LuriaBroth (10 g/L Trypticase-Peptone, 10 g/L yeast extract, and 5 g/L sodiumchloride). The culture was placed in a shaking platform incubator at 30°C. for 16 hours at 250 rpm. The culture was then transferred to 8 litersof sterile medium in a BioLafitte 15-L fermentor.

The 8 liters of medium that were sterilized in place in the fermentorconsisted of the following:

10 g/L yeast extract 5.25 g/L ammonium sulfate 3.5 g/L dibasic potassiumphosphate 4.0 g/L monobasic potassium phosphate 1.25 g/L sodium chloride

After the sterilized medium cooled to 30° C. the following was added:

40 g glucose 8 g magnesium sulfate-heptahydrate 16 ml trace metalssolution¹

The pH of the medium was then adjusted to 7.0 using concentratedphosphoric acid. The other parameters of the fermentation during thisbatch phase were set as follows:

-   -   air flow rate=31.0 L/min    -   agitation=350 rpm    -   dissolved oxygen readout set at 60%    -   oxygen flow rate=0    -   back pressure=0.5 bar

Once the culture in the fermentation vessel reached at O.D.600 of 6.0, aconcentrated solution of glucose and organic nitrogen was started usinga schedule that ramps the feed flow according to the O.D. of theculture. This concentrated feed (Feed 1) consisted of the following:

50 g/L Trypticase-peptone 50 g/L yeast extract 450 g/L glucose 8.5 g/LMagnesium-heptahydrate 10 ml trace metals solution¹ 10 ml vitaminsolution²

At the time that the concentrated feed was first introduced into thefermentor, the following changes were made:

agitation raised to 850 rpm

back pressure raised to 0.8 bar

Using the concentrated feed, the O.D. was increased to 30. At that pointthe culture was induced by raising the temperature to 42° C. Otherchanges were made as follows:

air flow rate decreased to 24 L/ min

oxygen flow rate increased to 3 L/min

feed 1 decreased to 0

feed 2 started at 300 ml/hr

Feed 2 consisted of the following:

200 g/L Trypticase-peptone

100 g/L yeast extract

110 g/L glucose

After 4 hours at 42° C. the fermentation was halted and the cells wereharvested by centrifugation into plastic bags contained within a oneliter centrifuge bottle. Centrifugation was at 400 rpm for 60 minutes.At the end of this period, the supernatant was removed and the remainingcell paste was frozen at −90° C.

¹Trace Metals Solution:

27 g/L FeCl₃ · 6H₂O 2 g/L ZnCl₂ · 4H₂O 2 g/L CaCl₂ · 6H₂O 2 g/L Na₂ ·MoO₄ · 2H₂O 1.9 g/L CuSO₄ · 5H₂O 0.5 g/L H₃BO₃ 100 ml/L concentrated HCl²Vitamin Solution:

0.42 g/L riboflavin 5.4 g/L pantothenic acid 6 g/L niacin 1.4 g/Lpyridoxine hydrochloride 0.06 g/L biotin 0.04 g/L folic acidNH₂-Terminal Amino Acid Sequencing

NH₂-terminal amino acid sequence of E. coli-derived recombinant TIMP-3protein was determined to be identical to the sequence deduced from thecDNA clones. The methionine initiator from the construct was cleavedoff. There was no other detected proteolytic processing at the TIMP-3NH₂-terminus. No assignment was made for cys-1 and cys-2 since theprotein sample was reduced and reduced cysteines cannot readily bedetected by this method. Therefore, the sequence read as follows:X-T-X-S-P-S-H-P-Q-D-A-F- (SEQ ID NO:41).

Methods

Partially purified recombinant TIMP-3 present in E. coli inclusionbodies was electrophoresed on an SDS polyacrylamide gel andelectroblotted onto a PVDF membrane for sequence analysis. NH₂-terminalamino acid analysis was performed on a gas-phase sequenator (model 477,Applied Biosystems, Foster City, Calif.) according to publishedprotocols. Hewick et al., J. Biol. Chem., 256: 2814–2818 (1981). Thesequenator was equipped with an on-line phenylthiohydantoin (PTH) aminoacid analyzer and a model 900 data analysis system (Hunkapiller et al.,Methods of Protein Microcharacterization, Clifton, N.J.: pp. 223–247(1986)). The PTH-amino acid analysis was performed with a micro liquidchromatography system (model 120) using dual syringe pumps and reversedphase (C-18) narrow bore columns (Applied Biosystems, Inc.), with thedimensions of 2.1 mm×240 mm.

Protein Purification

Approximately 435 g wet weight of E. coli cell paste, harvested from thefermentation run was resuspended to a volume of 1760 ml in water andbroken by two passes through a microfluidizer. The cell lysate wascentrifuged at 17,700×g for 30 min, and the pellet fraction was washedonce with water (by resuspension and by recentrifugation). A portion ofthe washed pellet material (3.1% of the total) was resuspended in 10 mlof 50 mM Tris-HCl/50 mM dithiothreitol/2% (w/v) sodiumN-lauroylsarcosine, pH 8.5. After incubation at 50° C. for 5 min, and atroom temperature for 3 hr, the mixture was centrifuged at 20,000×g for60 min. The supernatant was applied to a Sephacryl S-200 gel filtrationcolumn (Pharmacia; 2×23 cm) equilibrated in 20 mM Tris-HCl/1% sodiumN-lauroylsarcosine, pH 8.0, at room temperature. Fractions of 1 ml werecollected at a flow rate of 5 ml/hr and analyzed by A₂₈₀ and bySDS/polyacrylamide gel electrophoresis (PAGE). Fractions 43–53were-pooled, and the pool was dialyzed over a 3-day period against 20 mMTris-HCl (pH 8.0), 0.02 % (w/v) sodium azide, at 4° C.

FIG. 3 presents a silver stained SDS-PAGE gel of the partially purifiedexpression product from this fermentation. Lanes 3 and 4 contain reducedE. coli derived TIMP-3, pre- and post- dialysis. Lanes 9 and 10 containunreduced E. coli derived TIMP-3, pre- and post- dialysis. As can beseen, the apparent molecular weight for reduced material isapproximately 22 kDa.

As can be seen from FIG. 3, the post-dialysis yield was reduced; thepolypeptide appeared to be somewhat unamenable to solubilization. In thepresent process, the presence of inclusion bodies containing relativelyinsoluble material resulted in a reduced yield of purified and isolatedTIMP-3. Although this resulted in a partially purified product, oneskilled in the act will recognize methods to obtain a purified andisolated polypeptide. For example, one may use different detergents assolubilizing agents, or use a different expression system, for example,one which permits secretion of the polypeptide (and thus elimination ofinclusion bodies).

Expression and purification was also attempted using eucaryotic cells(COS-7 cells), however no active recombinant TIMP-3 polypeptide wasobserved. This may have been due to adherence of the recombinant TIMP-3polypeptide to extracellular matrix material produced by COS-7 cells.One possible way to obtain active protein from a mammalian host cell maybe to use cells which are non-adherent, and therefore produce nosignificant amount of extracellular matrix material. The recombinantpolypeptide would then be found in the conditioned culture medium. Forexample Jurkat cells or U937 cells may be used for recombinantpolypeptide expression, and other non-adherent host cells and expressionsystems will be apparent to those skilled in the art.

Results of Screening Two cDNA Libraries and Expression of RecombinantHuman TIMP-3

The work herein presents the cloning and expression of a third class ofmammalian TIMP family members, herein collectively referred to as“TIMP-3.” The nucleotide sequence obtained from a human fetal kidneycDNA library is presented in FIG. 1 (SEQ ID NO:12). As can be seen, thenucleotide sequence obtained contains 1240 base pairs. The predictedamino acid sequence is also presented (SEQ ID NO:13). (The amino acidsequence is predicted, as the polypeptide itself was not fullysequenced. One skilled in the art may sequence the expression product ofthe E. coli deposited at the ATCC, accession no. 69455.) The predictedinitial cysteine of the mature protein is number +1. The prediction isbased upon comparison to other members of the TIMP family.

FIG. 4 presents this comparison among the known members of the TIMPfamily. Bullet points (•) indicate those amino acid residue which areunique to the TIMP-3 of FIG. 1 obtained from expression of human cDNA,and bold-face type indicates conserved residues.

As can be see, the present human recombinant TIMP-3 of FIG. 1 isdistinct from all other members of the TIMP family. While possessing theconserved cysteine residues and other conserved amino acids within thefamily (39, total), at least 23 amino acid residues are unique to theillustrated human recombinant TIMP-3.

FIGS. 5–13 illustrate the differences between the present humanrecombinant TIMP-3 of FIG. 1 and chicken TIMP-3 (“ChIMP-3,” FIGS. 5–7),human TIMP-2 (FIGS. 8–10), and human TIMP-1 (FIGS. 11–13), at both theamino acid and nucleic acid levels. The Figures contain a solid linebetween amino acid residues which are identical, and dots indicating thedegree of evolutionary distance. (For FIGS. 5, 8, and 11, illustratingamino acid alignment, the numbering at position “1” is for the maturepolypeptide.)

At the amino acid level, TIMP-3 and ChIMP-3 are approximately 80%identical, with identical amino acids being more or less disperseddiscontinuously, (FIG. 5). FIG. 6 shows that, at the nucleic acid level,FIG. 1 TIMP-3 DNA is approximately 74% homologous with ChIMP-3 DNA,between nucleic acids 151–1087 (TIMP-3) and 1–886 (ChIMP-3). FIG. 7shows that even analyzing the region of maximal homology, base pairs282–1040 from FIG. 1 TIMP-3, and 113–884 for ChIMP-3), there isapproximately 78% identity.

FIGS. 8–10 illustrate a comparison between human recombinant TIMP-3 ofFIG. 1 and human TIMP-2. At both the amino acid level and the nucleicacid level, there are greater distinctions than with ChIMP-3. FIG. 8shows that there is approximately 46% identity at the amino acid level.FIG. 9 shows that, at the nucleic acid level, the overall homology isapproximately 52% overall, and approximately 60% in the region ofmaximal homology (FIG. 10).

FIGS. 11–13 illustrate a comparison between human recombinant TIMP-3 ofFIG. 1 and human TIMP-1. At the amino acid level, there is approximately39% identity (FIG. 11), and approximately 47% overall homology at thenucleic acid level. There is approximately 65% identity in the region ofmaximal homology.

Biochemically, the calculated isoelectric points (pI) of the matureTIMP-3 polypeptide and its pre-cursor are 9.16 and 8.80, respectively.There is a potential glycosylation site at the carboxy-terminal sequence(184:NAT). While naturally occurring ChIMP-3 is reported to benon-glycosylated (Pavloff et al., supra, J. Biol. Chem. 267: at 17323),it is not currently known whether naturally occurring human TIMP-3 isglycosylated. Regardless, the present invention includes polypeptideswith additional chemical moieties, such as carbohydrates. Thehydrophobic leader of the FIG. 1 material is 23 amino acids long.Sequencing of the N-terminus confirmed the identity of the first 12amino acids of the mature recombinant polypeptide.

The cloning and expression described herein demonstrates that thepresent TIMP-3 polypeptides represent new members in the TIMP family.

EXAMPLE 2

Expression of TIMP-3 in Various Cell Types

A variety of cells were tested for the expression of TIMP-3 RNA (whichwould indicate polypeptide expression). The results show that amongnormal (i.e., non-cancerous) cells, expression is observed in cellsassociated with extracellular matrix activity (i.e., growth ofdegradation). The normal cells (or tissues) where TIMP-3 RNA expressionwas seen (FIGS. 14A and B) are placenta, stromal cells, embryonic lung,newborn foreskin (one of two samples being slightly positive), and(slightly positive) adult lung. Among the cancer cells tested, some werepositive, some were negative. For example, various breast adenocarinomacell lines yielded disparate results; with one was positive, one wasnegative, one was slightly positive. This may indicate temporalexpression, in that TIMP-3 expression may vary over the course ofdisease progression, although the significance is unclear. Table 2,below, presents a description of the cells tested and the results. Beloware the methods.

In many of the positive cell lines three mRNA bands of approximate 2.2,2.5 and 4.4 kb size were detected. The significance of the differentmRNA bands is unknown but may represent alternative splicing or extended3′ or 5′ untranslated regions. These may be RNAs encoding differentnaturally occurring variants.

TABLE 2 ATCC Numbers Plus Description ATCC cell line ATCC NumberDescription Poly A northern Total RNA northern Hs 294T HTB 140metastatic melanoma strongly positive strongly positive HepG2 HB 8065hepatocellular carcinoma slightly positive A-704 plus or minus HTB 45adenocarcinoma, kidney negative HuT 78 TIB 161 T cell lymphoma negativeMCF-7 plus HTB 22 breast adenocarcinoma slightly positive MCF-7 minusHTB 22 breast adenocarcinoma negative MDA-MB-231 HTB 26 breastadenocarcinoma positive MDA-MB-453 HTB 131 breast carcinoma negative Hs68 CRL 1635 newborn human foreskin slightly positive Hs 27 CRL 1634newborn human foreskin negative A 172 CRL 1620 glioblastoma negative Hs578 T HTB 126 ductal carcinoma, breast strongly positive A-498 HTB 44carcinoma, kidney borderline positive 293 CRL 1573 transformed embryonalborderline positive kidney SK-NEP-1 HTB 48 Wilms' tumor (kidney)borderline positive WI-38 CCL 75 normal embryonic lung positive WI-26VA4 CCL 95.1 SV40 virus transformed borderline positive lung CCD-11LuCCL 202 normal lung borderline positive DU 4475 HTB 123 breastcarcinoma, metastatic negative nodule BT-474 HTB 20 ductal carcinoma,breast negative Caov-3 HTB 75 adenocarcinoma, ovary slightly positiveSK-OV-3 HTB 77 adencarcinoma, ovary negative SK-Hep-1 HTB 52adenocarcinoma, liver slightly positiveMethods

Two types of Northern blots were performed, one on total RNAtranscripts, and one using poly A+tailed transcripts.

Total RNA Preparation. Total RNA for the total RNA northern wasextracted from cells using a modification of a published protocol(Chomczynski and Sacchi, Anal. Biochem. 162: 156–159 (1987).

Cells grown in 2×10 cm petri dishes (approximately 2×10⁶ cells), werewashed two times with cold 1×PBS. After all of the PBS was aspiratedoff, 500 μl of an aqueous solution containing the following was added toeach dish: 4 M guanidinium thiocyanate (Fluka), 25 mM sodium citrate pH7.0 (Mallinckrodt), 0.5% sarcosyl (Sigma, St. Louis, Mo.) 0.1Mβ-mercaptoethanol (Sigma, St. Louis, Mo.). The cell lysate was pipettedinto a 1.5 ml Eppendorf microfuge tube and was sheared with a 25 gaugeneedle.

Sodium acetate (pH 4) was added to the 500 μl lysate to make a finalconcentration of 0.2 M. The mixture was shaken vigorously by hand. ⅕volume of chloroform was added and mixed thoroughly. The tubes were spunat 15,000 rpm for 5 minutes at 20° C. in a Tomy MTX-100 centrifuge. Thetubes were inverted to allow the white precipitate layer to separatefrom the aqueous layer instead of respinning. The RNA was re-extractedwith phenol and chloroform two additional times and was extracted onefinal time with chloroform. 1 ml of isopropanol was added to themicrofuge tube and the mixture was precipitated at −20° C. overnight.The next day it was spun at 15,000 rpm for 15 minutes. The pellet waswashed with 1 volume of 80% ethanol, re-spun, and dried in a Speed Vac(Savant, Farmingdale, N.Y.).

The pellet was resuspended in 400 μl of the guanidinium solution whichcontained β-mercaptoethanol (Sigma, St. Louis, Mo.). 800 μl of ethanolwas added to this mixture, which was then spun at 15,000 rpm for 15minutes and washed with 80% ethanol. This pellet was resuspended in 20μl of water and the O.D. was determined.

Poly A+RNA Preparation. Poly A+RNA was prepared using Clontech (PaloAlto, Calif.) oligo dT-cellulose spun columns. 2×1 ml of a high saltbuffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 0.5 M NaCl) was washedthrough the columns and drained by gravity. Total RNA, isolated asdescribed above, was resuspended in 1 ml of elution buffer (10 mMTris-HCl [pH 7.4], 1 mM EDTA) and was heated at 68° C. for 3 minutes.0.2 ml of sample buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 3M NaCl)was added to the RNA solution, which was then placed on ice.

The samples were loaded onto the freshly equilibrated columns andallowed to soak under gravity. The columns were placed inside 50 mltubes and were centrifuged at 350×g for 2 minutes. The eluates werediscarded. 0.25 ml of the high salt buffer (see above) was added to eachcolumn and the columns were centrifuged at 350×g for 2 minutes. Thiswash was repeated once. In each case, the eluates were discarded. Thecolumns were then washed 3 times with low salt buffer (10 mM Tris-HCl[pH 7.4], 1 mM EDTA, 0.1 M NaCl) and centrifuged each time at 350×g for2 minutes. The eluates were discarded in each instance. Sterile 1.5 mlmicrocentrifuge tubes were placed inside of the 50 ml tubes to collectsubsequent elutions. 0.25 ml of elution buffer (10 mM Tris-HCl [pH 7.4],1 mM EDTA,) warmed to 65° C. were applied to the columns, which werethen spun at 350×g for 2 minutes. This procedure was repeated 3 timesfor a total of 4 elutions per column. For each column, all of theelutions were collected in a microcentrifuge tube. The eluents wereethanol precipitated as above.

Northern Blotting. 10 μg of total RNA was loaded in each lane. Thesample buffer included 10 μl of formamide, 3.5 μl of formaldehyde, 2 μlof 10×MOPS, 2 μl of loading dye, 0.2 μl of ethidium bromide, and 6.5 μlof RNA sample in water. The poly A+RNA blot had 3 μg of mRNA loaded ineach lane.

The gels for the northern blots consisted of 1.5 g of agarose melted in95 ml of water and then cooled to 60° C. 30 ml of 5×MOPS and 25 ml offormaldehyde (pH 4.7) were added to the cooling agarose solution. Priorto transfer, the gels were trimmed to remove excess gel. They were thensoaked in distilled water for 5 minutes, followed by a 10 minute soak in50 mM NaOH, 10 mM NaCl at room temperature. The gels were neutralized in0.1 M Tris-HCl, pH 7.5 for 45 minutes and then soaked in 20×SSC for 1hour. Transfer occurred overnight in 10×SSC. The gels were blotted ontoSchleicher & Scheull (Keene, N.H.) nitrocellulose membranes. The totalRNA gel was blotted onto pure nitrocellulose and fixed by UVcrosslinking using a Stratalinker (Stratagene, La Jolla, Calif.). Thepoly A+ gel was blotted onto supported nitrocellulose and was baked in avacuum oven for 2 hours at 80° C.

The blots were hybridized in a manner similar to the screening of thecDNA library. The sole difference is that for the northern blotanalysis, RNase-free reagents were used wherever possible.

EXAMPLE 3

In Vitro Activity of Human Recombinant TIMP-3

Modified Zymogram

DeClerck et al. J. Biol. Chem. 2: 17445–17453 (1991) showed that TIMP-2will bind to pAPMA-activated rabbit fibroblast interstitial collagenasein complexes that are stable in SDS. The 52 kDa inactive presursor wascleaved to an active 42 kDa protein by the organomercurial. Although theactive protein primarily degrades type I, II and III collagen, it willalso degrade gelatin to a lesser degree.

Conditioned medium (CM) from rabbit synovial fibroblasts containsinterstitial collagenase as well as 72 kDa type IV gelatinase. The CMwas incubated in 5 μl of 50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl₂, pH7.5 for 15 minutes in either the presence or absence of TIMP-2(according to EP 0 398 753), TIMP-2Δ or the FIG. 1 TIMP-3. Note thatTIMP-2Δ refers to a trucated biologically active form of TIMP-2 withamino acids 128–194 of the mature protein deleted. Tolley et al., J.Mol. Biol. 229: 1163–1164(1993); Willenbrock et al., Biochemistry 32:4430–4437 (1993). It has previously been shown that TIMP-2 interactspreferentially with 72 kDa procollagenase but that these complexes werenot stable in 0.1% (w/v) SDS. Stetler-Stevenson, J. Biol. Chem., 264:17374–17378 (1989). The TIMP-3 tested was the dialyzed TIMP-3 of FIG. 3.

In the absence of TIMPs, 2 zones of clearing are visible when CM fromrabbit synovial fibroblasts is run on a 10% acrylamide, 0.1% gelatingel. FIG. 15. One of the bands corresponds to 42 kDa pAPMA-activatedinterstitial collagenase. This clearing was absent in the presence of CMincubated with TIMP-2, TIMP2Δ, or the FIG. 1 TIMP-3. The other zone ofclearing was not affected, meaning that it did not form as SDS-stablecomplex with the TIMP. In a separate experiment using the presentmethods (data not shown) a zone of clearing generated by the collagenasein medium conditioned by COS-7 cells was not inhibited by the presenceof TIMP-2, TIMP-2Δ or TIMP-3.

EXAMPLE 4

Preparation of TIMP-3 Polypeptide Analogs and Nucleic Acid Variants

The amino acid sequence of full length TIMP-3 is presented in FIG. 1.Using the numbering of FIG. 1, the full length sequence is 188 aminoacids long. The amino acid sequence at −23 through −1 is a leadersequences and thus the pro version of the polypeptide is 211 amino acidsin length.

The coding region of the TIMP-3 DNA of FIG. 1 is −69 through position564 of the nucleic acid sequence illustrated.

Alternatively, for either variant, one may construct a signal peptidesequence for eucaryotic cell expression. As can be seen from FIG. 16,two additional cDNA clones have been isolated, TIMP3clone2 (SEQ IDNOs:14, 15, ATOC Accession No. 69456) and TIMP3HCM-3 (SEQ ID NOs:16, 17,ATCC Accession No. 69453). These clones represent natura variants.TIMP3clone2 lacks part of the region encoding the N-terminus of theleader sequence of TIMP3clone7. As such, this would be preferablyexpressed in a procaryote, such as E. coli. TIMP3HCM-3 lacks a portionof the region encoding the NH₂-terminus of the mature protein. Sincethis clone lacks the hydrophobic leader sequence, it would be preferablyexpressed in a procaryote, such as E. coli.

FIG. 16 shows that there are some differences among the three cDNAclones. At nucleotide 320, there is an A in TIMP3clone 2 and a T inTIMP3clone 7. This would result in a change in the amino acid sequencefrom a trp to an arg at position 14 in the hydrophobic leader sequence.This difference may be a cloning artifact due to its location at the 5′end of that clone. ChIMP-3 also has a trp at this position. Anotherdivergence can be found at base 529, in which clone 2 has a C and clones7 and HCM-3 have a T. This polymorphism does not result in an amino acidchange because both CAT and CAC encode his. Other polymorphisms arefound in or near the poly A tail. The poly A tail of HCM-3 is precededby a single G, whereas in the other 2 clones it is preceded by GG. Thepoly A tail of clone 7 is 15 bases long and the poly A tail of HCM-3 is18 bases long. The poly A tail of clone 2 is 17 bases long, isinterrupted by 3 other bases, and is followed by 32 nucleotides ofadditional 5′ untranslated sequence.

PCR product 29 (TIMP3PCR29 SEQ ID NOs:18, 19 see FIG. 16) was alsoobtained from the human fetal kidney cDNA screening, using one insertspecific primer and one vector specific primer as follows: SEQ ID NO: 21(496-16) (CLWTDM forward):

-   5′-CGG AAT TCT GTC TCT GGA CCG ACA TGC TCT CC-3′ SEQ ID NO:20    (489-23) (lambda gt11 reverse):-   5′-GAC ACC AGA CCA ACT GGT AAT G-3′

As can be seen from FIG. 16, this may represent a naturally occurringC-terminal variant. At FIGS. 16B, bottom, to 16C, top, differences inamino acid sequence between TIMP3clone7 and TIMP3PCR29 are indicated.TIMP3PCR29, cloned into pUC19 and placed into E. coli has been depositedat the ATCC with accession no. 69532. A full cDNA clone encompassingthis PCR product has not been found in the fetal kidney cDNA library,however. It is unknown if TIMP3PCR29 represents a full or partialvariant or a PCR artifact.

Other TIMP-3 analogs may be prepared. One type of analog is a truncatedform which exhibits binding to the portion of a metalloproteinase whichbinds zinc. As indicated supra, the conserved region for this zincbinding domain may be represented by H E X G H, wherein X is either F orL. By analogy to TIMP-2 deletion analogs which have been prepared,TIMP-3 analogs maintaining enzyme inhibition activity may also beprepared.

FIG. 17 is an illustration of the proposed secondary structure for theTIMP family of proteins. See Alexander et al., Extracellular MatrixDegradation, in, Cell Biology of Extracellular Matrix (2d ed., Hay,ed.), Plenum Press, New York, pp. 255–302. As can be seen, the sixC-terminal cysteines form a secondary structure which is somewhatseparate from the structure formed by the region encompassing the firstsix cysteines. Previously, TIMP-2 analogs lacking the C-terminus up toand including the 6th cysteine in from the C-terminus have been shown tohave activity. Willenbrock et al., Biochemistry 32: 4330–4337 (1993).TIMP-3 analogs lacking one or more of the C-terminal cysteines are thosehaving the sequence (referring to the numbering of FIG. 1) of 1–121, andany of 1–122 through 1–188. Additions, deletions, and substitutions mayalso be made to amino acids 122–188, as well as attachment of chemicalmoieties, such as polymers.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

1. A composition comprising an isolated DNA sequence encoding a humanrecombinant tissue inhibitor of metalloproteinase 3 polypeptide(recombinant TIMP-3 polypepetide) and a pharmaceutically acceptablecarrier, wherein the recombinant TIMP-3 polypeptide is: (a) apolypeptide comprising the amino acid sequence 1 to 211 of SEQ ID NO:13; (b) a polypeptide comprising the amino acid sequence 24 to 211 ofSEQ ID NO: 13; (c) a polypeptide comprising the amino acid sequence 24to 144 of SEQ ID NO: 13 and, optionally, at the C-terminus, all or partof the amino acid sequence 145–211 of SEQ ID NO: 13; or (d) apolypeptide according to (b) or (c) additionally comprising a methionylresidue at position −1.
 2. The composition of claim 1 wherein saidcarrier is selected from the group consisting of a lipid solutioncarrier, a liposome, and a polypeptide.
 3. A composition comprising anisolated DNA sequence according to SEQ ID NO: 12 and a pharmaceuticallyacceptable carrier, wherein the DNA sequence encodes a polypeptidecomprising the amino acid sequence 24 to 211 of SEQ ID NO: 13 and,optionally, a methionyl residue at position −1.
 4. The compositionaccording to claim 3 wherein said carrier is selected from the groupconsisting of a lipid solution carrier, a liposome, and a polypeptide.5. An antisense DNA according to all or part of SEQ ID NO: 12, whereinthe antisense DNA modulates or prevents expression of endogenous humanTIMP-3 nucleic acids.
 6. An article of manufacture comprising apackaging material and a pharmaceutical agent, wherein saidpharmaceutical agent contains a DNA encoding human TIMP-3, wherein theDNA encoding human TIMP-3 comprises a nucleic acid sequence selectedfrom SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, and wherein saidpackaging material comprises a label which indicates that saidpharmaceutical composition may be used for an indication benefiting fromgenetic therapy using such DNA.
 7. The article of manufacture accordingto claim 6 wherein said indication is emphysema.
 8. A kit comprising aDNA encoding human TIMP-3 and one or more additional factors affectingthe ex vivo growth of cells transformed or transfected with said DNA,wherein the DNA encoding human TIMP-3 comprises a nucleic acid sequenceselected from SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, andwherein the one or more additional factors is selected from cytokinesand growth factors.
 9. The kit according to claim 8, wherein the kitadditionally comprises stem cell factor (SCF).